Herpes simplex virus 1 accelerates the progression of Alzheimer’s disease by modulating microglial phagocytosis and activating NLRP3 pathway

Accumulating evidence implicates that herpes simplex virus type 1 (HSV-1) has been linked to the development and progression of Alzheimer’s disease (AD). HSV-1 infection induces β-amyloid (Aβ) deposition in vitro and in vivo, but the effect and precise mechanism remain elusive. Here, we show that HSV-1 infection of the brains of transgenic 5xFAD mice resulted in accelerated Aβ deposition, gliosis, and cognitive dysfunction. We demonstrate that HSV-1 infection induced the recruitment of microglia to the viral core to trigger microglial phagocytosis of HSV-GFP-positive neuronal cells. In addition, we reveal that the NLRP3 inflammasome pathway induced by HSV-1 infection played a crucial role in Aβ deposition and the progression of AD caused by HSV-1 infection. Blockade of the NLRP3 inflammasome signaling reduces Aβ deposition and alleviates cognitive decline in 5xFAD mice after HSV-1 infection. Our findings support the notion that HSV-1 infection is a key factor in the etiology of AD, demonstrating that NLRP3 inflammasome activation functions in the interface of HSV-1 infection and Aβ deposition in AD. Supplementary Information The online version contains supplementary material available at 10.1186/s12974-024-03166-9.


Introduction
Alzheimer's disease (AD) is the most common neurodegenerative disorder, resulting in progressive memory loss and dementia.It exists in two forms: a genetic etiology of early-onset familial AD (fAD) caused by mutations of genes, such as APP, PSEN1 and PSEN2 genes; and a sporadic form of late-onset AD (LOAD) caused by a complex multifactorial etiology [1][2][3].The deposition of senile plaques enriched with β-amyloid (Aβ) peptides is a pathological hallmark of both fAD and LOAD [4].Aβ is primarily produced in neurons through the sequential processing of APP by β-secretase/ BACE1 and γ-secretase/Presenilin [5,6].Traditionally, Aβ has been identified as the primary cause of the disease in fAD, known as the "Aβ cascade hypothesis", where increased production of toxic Aβ species initiates a series of progressive changes that ultimately lead to neurodegeneration [7,8].However, the precise mechanism of Aβ production and clearance in LOAD is not clear.
Mounting evidence suggests that pathogens are involved in the progression of AD pathogenesis [9][10][11].The AD pathogen hypothesis suggests that viral or microbial infection of the central nervous system (CNS) may act as triggers to induce a pathological cascade, leading to accumulation of Aβ [12][13][14].Several pathogens, including Chlamydophila pneumoniae [15], Borrelia spirochetes [16], Candida glabrata [17], human herpesviruses 6 and 7 (HHV6 and HHV7) [13] and herpes simplex virus 1 (HSV-1) [11,18,19], have been linked to LOAD.Among these pathogens, HSV-1 has emerged as one of the leading pathogens and has been proposed as a potential risk factor in AD pathogenesis [20,21].Independent cohort studies have revealed that HSV-1 is strongly associated with neurodegenerative diseases, especially AD [20,22].It has been established that HSV-1 is present in the brains of a high proportion of elderly normal subjects and Alzheimer's disease (AD) patients [23,24].
HSV-1 is a neurotropic double-strand DNA virus that establishes a latent infection in the sensory neurons of trigeminal ganglia of humans [25].HSV-1 brain infection leads to the activation of glial cells, especially microglia cells [26].Microglia cells, the resident innate immune cells in the central nervous system, play essential roles in healthy and neurodegenerative diseases [27].In physiological conditions, resting microglia have ramified processes that constantly survey the microenvironment, assisting neuronal development, synaptic pruning and support neuronal survival [28,29].In response to pathogen infections or neurodegenerative disorders, reactive microglia mediate phagocytic uptake and the secretion of inflammatory cytokines and chemokines [30][31][32].HSV-1 infection activates the cGAS-STING signaling pathway and NLRP3 inflammasome pathway in microglia to fight against viral invsion [33][34][35][36].In the other hands, the pathogen-mediated innate immune response also could cause neuroinflammation, which is an important hallmark of neurodegenerative diseases.However, the mechanism of microglia activation and innate immune signaling in AD pathology upon HSV-1 infection remains largely unclear.Therefore, a comprehensive study of microglia, Aβ deposition, and neuroinflammation after HSV-1 infection might deepen our understanding of AD.
In this study, we administrated HSV-1-GFP into 5xFAD mice and investigated the effects of HSV-1 infection on Aβ deposition and cognitive function.The results revealed that HSV-1 infection led to Aβ deposition and cognitive deficits in 5xFAD mice.Following HSV-1 infection, microglia were recruited to the viral core but not Aβ deposition, enhancing their phagocytic uptake of viruses, thereby leading to the accumulation and deposition of Aβ.Moreover, the NLRP3 inflammasome signaling was activated after HSV-1 infection and drove the accumulation of Aβ aggregates.Administration of MCC950 sodium, a selective small-molecular inhibitor of the NLRP3 inflammasome, reduced Aβ deposition and ameliorated cognitive decline in 5xFAD mice following HSV-1 infection.Together, our findings support the concept that HSV-1 infection and NLRP3 inflammasome pathway are connected to the development and progression of AD pathology.

Transgenic mice and mouse infection model
The 5xFAD transgenic mice (B6SJL-Tg (APPSwFlLon, PSEN1*M146L*L286V) 6799Vas/Mmjax, 34840-JAX) were originally obtained from the Jackson laboratory and maintained on the C57BL/6 background.For behavioral tests, 3-month-old male 5xFAD mice were used.For other in vivo experiments, age and sex-matched mice were used.Mice were housed with free access to water and food under a 12 h/12 h light-dark cycle.All experimental animal procedures were approved by the Institutional Animal Care and Use Committee of Tsinghua University.
For behavioral tests, 3-month-old male 5xFAD mice received bilateral injections of HSV-1-GFP viron suspension at coordinates AP, − 2.0; ML, ± 1.5; DV, − 2.0.This injection was carried out using a 5 μL syringe equipped with a 30-gauge needle attached to a digital stereotaxic apparatus and an infusion pump, administrated at a rate of 0.2 μL/min.Following the completion of each injection, the needle was retained in place for 5 min before gradual withdrawal.21 days after the injections, the mice were subjected to a battery of behavioral tests.
In the case of other in vivo experiments, we employed the approach involved by Eimer and coauthors [13].In brief, age and sex-matched 5xFAD mice received unilateral injections of HSV-1-GFP viron suspension at the coordinates AP, − 2.0; ML, − 1.5; DV, − 2.0 (right side) using a 5 μL syringe fitted with a 30-gauge needle attached to a digital stereotaxic apparatus and an infusion pump, administrated at a rate of 0.2 μL/min.Sterile PBS was injected into the contralateral side of the brain as controls (left side).Subsequently, the mice were either perfused for immunostaining or euthanized for western blotting at the indicated time points.
Primary microglial cells were derived from P0-P5 WT mice as previously described [4,37].Briefly, neonatal cortex was collected, trypsinized for 10 min, and filtered through a 70 μm filter.Cells were then cultured in DMEM medium containing 10% FBS and 1% penicillin/ streptomycin for 2-3 weeks.The medium was replaced with fresh cell culture medium every three days.The microglia were isolated by shaking (200-220 rpm, 3 h) after 2-3 weeks of primary cultivation.The medium containing detached microglia was collected and seeded into PDL-coated 12-well or 24-well plates.After the cells attached, the medium was replaced with fresh cell culture medium.

Virus production
Herpes simplex virus 1-GFP (HSV-1-GFP, KOS Strain, generously provided by Dr. Daxing Gao, University of Science and Technology of China), was employed in the innate immune studies [38].The virus was amplified in Vero cells as described in previous paper [39,40].The supernatant containing the virus was harvested and centrifuged at 5000 rpm/4 °C.The viral suspension was filtered through a 0.22 μm filter and purified by ultracentrifugation as 20,000 rpm/2 h.The concentrated virus was aliquoted and stored at − 80 °C.
For the Aβ and HSV-GFP phagocytosis in vitro assay, HT22 cells were infected with HSV-1-GFP for 24 h, then washed and suspended with PBS.BV2 and primary microglial cells were incubated with 1 μg/ml Alexa 555-labeled Aβ 1-42 (Anaspec), or 1 μg/ml Alexa 555-labeled Aβ 1-42 plus HSV-1-GFP from HT-22 neuronal cells for 4 h.Subsequently, cells were fixed with 4% paraformaldehyde for 20 min at room temperature and stained with DAPI.The cells were then imaged with the Zeiss LSM780.

Thioflavin S staining
Thioflavin S staining was used to label the Aβ plaques.Briefly, brain sections were stained with 0.1% Thioflavin S (Thio-S, sigma) in the dark for 8 min in 50% ethanol, followed by two washes with 50% ethanol and three washes with PBS.Subsequently, the sections were mounted for imaging.

Microscopy and image analysis
All imaging was performed using an OLYMPUS FV3000RS-BX and ZEISS 780 Confocal laser scanning microscope with × 4, × 10, × 20, × 40 and × 63 objectives.For Aβ plaques quantification, ten views from the hippocampus of five mice and the entire brain were assessed.For microglia, astrocyte and inflammasome markers quantification, Iba1-, GFAP-, CD68-, ASC-, and NLRP3-positive cells were counted and then calculated as the Iba1 + , GFAP + , CD68 + , ASC + and NLRP3 + cells divided by DAPI cells.To assess Aβ plaque and HSV-GFP phagocytosis by microglia cells, confocal single-plane images of lysosomes (CD68), Aβ and microglia (Iba1) were separately isolated, and the areas of colocalization (CD68 + Aβ + GFP + or Iba1 + Aβ + GFP + ) were measured by ImageJ software.Image processing and analysis were performed by Image J software or Imaris software (Bitplane, Switzerland), as appropriate.Imaris were used for 3D reconstruction of confocal images to analyze microglia morphology and colocalization of Aβ plaque with CD68, GFP with CD68, Aβ plaque with Iba1, and GFP with Iba1.

RNA isolation and quantitative real-time PCR (qRT-PCR)
PBS or HSV-GFP (1 × 10 5 PFUs/hippocampus) was administered into 3-month-old 5xFAD mice.Three and seven days after HSV-1 infection, control and HSV-1-infected hippocampi from the same mice were collected and homogenized in TRIZOL reagent to isolate total RNA.Subsequently, cDNA was reverse transcribed using the One-Step gDNA Removal and cDNA Synthesis Kit (Mei5Bio).Relative mRNA expression of IL6, IL-1β, TNFα, IFNβ, IFNγ, IL10, and CCL5 was determined by quantitative real-time PCR.

Behavioral tests
Mice were housed in groups of 3-5 animals on a 12:12 h light-dark cycle.3-month-old male 5xFAD mice, which were infected with sterile PBS or HSV-1-GFP, were used for all the behavioral tests.Videos were recorded and analyzed by the Smart V3.0.03 software (Panlab, Barcelona, Spain).

Y-maze test
Y-maze spontaneous alteration test was carried out to assess short-term spatial memory.Mice were allowed to acclimate to the testing room for 48 h.The Y-maze apparatus comprised three opaque arms at 120° angles from each other.Each mouse was gently placed in the distal part of the maze and allowed to explore freely for 5 min.Detailed recordings were made of their movements and the total number of arms entered.Spontaneous Alteration [%] was defined as the ratio of consecutive entries into 3 different arms divided by the total arm entries minus 2.

Morris water maze test
A circular water tank (diameter: 120 cm) was filled with water to a depth of 25 cm, and the water was made opaque with non-toxic white paint.A 13-cm in diameter round platform was hidden 1 cm beneath the surface of the water at the center of a given quadrant of the water tank.Mice received training for five consecutive days, with each session comprising four trails starting from different sites.For each trail, the mouse was gently released from the wall of the tank and allowed to explore, locate and stand on the platform for a duration of 20 s, within the 60-s trail period.Following the completion of training, a probe test was performed 24 h later.During the probe test, the platform was removed and task performance including swimming tracks, speed, time spent and entries into the platform were recorded for analysis.

Drug treatment
PLX3397 (Selleck, Cat# S7818) was employed for the pharmacological depletion of brain microglia cells.Briefly, mice were fed with PLX3397-formulated AIN-76A diet (290 mg/kg) or normal AIN-76A diet ad libitum [41], and the treatment continued until the mice were euthanized.

HSV-1 infection exacerbates Aβ plaque deposition and cognitive decline
To investigate the correlation between HSV infection and Aβ plaque deposition, we administrated HSV-1-GFP virus into the hippocampus of 3-month-old transgenic AD (5xFAD) mice, using phosphate-buffered saline (PBS) as a vehicle control (Fig. 1A).Seven days after stereotaxic injection of HSV-1-GFP, results of Aβ immunostaining revealed a dramatic increase of amyloid plaques in the whole brain (Fig. 1B and C).Thio-S staining of brain slices confirmed a significant increase in both the number and size of Aβ plaques in 3-month-old 5xFAD mice infected with HSV-GFP compared to those injected with PBS (Fig. 1D and E).The Aβ protein amounts in the hippocampus of 3-monthold 5xFAD-HSV mice were notably higher compared to 3-month-old 5xFAD-Ctrl mice (Fig. 1F).Accumulation and deposition of Aβ may trigger neurotoxic effects, neuronal loss, and degeneration, ultimately leading to cognitive impairments.To assess the detrimental effects of HSV-1 infection on amyloid deposition and its association with cognitive dysfunction in 5xFAD mice, we performed Y-maze and Morris water-maze tests in 3-month-old 5xFAD mice following virus injection.At 3 months of age, 5xFAD mice did not exhibit significant deficits in spatial learning and memory (Fig. 1G-I).However, HSV-1 infection resulted in significant impairments in spatial learning and memory, characterized by reduced spontaneous alterations without a decrease in total arm entries (Fig. 1G), prolonged escape latency time during the training trails and the probe trail, and a lower number of platform crossings (Fig. 1H and I).Swimming speeds were comparable across all groups of animals, thus excluding impairments in motor function (Fig. 1I).
To further confirm that HSV-1 facilitate Aβ accumulation, we also administrated HSV-1-GFP virus into the hippocampus of 1-month-old and 6-month-old 5xFAD mice and observed a significant increase in the number and size of Aβ plaques in 1-month-old 5xFAD-HSV mice (Fig. 2A and B) and 6-month-old 5xFAD-HSV mice (Fig. 2C and D).Of note, Aβ deposition was already clearly detectable in 6-month-old 5xFAD-Ctrl mice, whereas the density and signal intensity of Aβ plaques were stronger in 6-month-old 5xFAD-HSV mice (Fig. 2E  and F).The Aβ protein amounts in the hippocampus also increased in both 1-month-old 5xFAD-HSV mice (Fig. 2G) and 6-month-old 5xFAD-HSV mice (Fig. 2H).Together, these data indicate that HSV-1 infection can exacerbate β-amyloid deposition and contribute to spatial and learning memory deficits in 5xFAD mice.
(See figure on next page.)Fig. 1 HSV-1 infection exacerbates Aβ deposition and cognitive decline in 5xFAD mice.A Three-month-old 5xFAD mice were injected with 1 × 10 5 PFUs of HSV-GFP or PBS in the hippocampus of the brain.B Representative images of Aβ (MAOB2, red) in the whole brain of 3-month-old 5xFAD mice seven days after HSV-GFP infection.Scale bars, 200 μm, 100 μm and 50 μm.C Quantification of Aβ plaques and plaque area in the brain of 3-month-old 5xFAD mice infected with PBS or HSV-GFP.(n = 5 mice per group).D Representative images of Thioflavin S staining (gray) in the whole brain of 3-month-old 5xFAD mice seven days after HSV-GFP infection.Scale bars, 200 μm, 100 μm and 50 μm.E Quantification of Aβ plaques and plaque area in the brain of 3-month-old 5xFAD mice infected with PBS or HSV-GFP.(n = 5 mice per group).F Three-month-old 5xFAD mice were randomly divided to three groups: control group (no treatment); Vehicle group (PBS treatment) and HSV-GFP group.Mouse hippocampi were collected and homogenized for immunoblotting of Aβ seven days post-infection.The intensities of Aβ were quantified by ImageJ software.G Three-month-old 5xFAD mice were randomly divided to two groups: control group (PBS treatment) and HSV-GFP group.Y-maze test was performed to measure the total number of arm entries and spontaneous alteration (%) at 21 days post-infection.(n = 9 mice per group).H, I Morris water maze was performed to evaluate the spatial learning and memory of 5xFAD mice infected with HSV-GFP.During the training phase, each group showed improved latency to the platform, but compared with the control group, the HSV-GFP group exhibited a significant delay to locate the platform (H).In the probe test, there was no significant difference in swimming speeds between the two groups of the mice.Compared to the control group, the 5xFAD-HSV group exhibited a significantly longer latency to locate the platform, but fewer target crossings (I).(n = 9 mice per group).Data are presented as means ± SEM. n.s.: p > 0.05, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001

HSV-1 infection promotes microglia cell activation in 5xFAD mice
The proliferation and activation of glial cells are prominent features of virus infection [42,43].Iba1 and GFAP are widely used as immunohistochemical markers for microglia and astrocytes, and their expression increases upon glial cell activation.Indeed, we observed increasing expression of Iba1 and GFAP in the hippocampus of 5xFAD mice infected with HSV-1, regardless of their age (Fig. 3).Interestingly, we observed distinct microglia morphology that may represent different activation states following HSV-1 infection.In the viral core, microglia often displayed an activated "rounded" morphology, while in the periphery of the viral core, microglia displayed an activated "ameboid" morphology characterized by shorter processes, larger cell bodies and reduced microglia volumes (Fig. 4A).In contrast, microglia exhibited a ramified morphology in 5xFAD-Ctrl mice (Fig. 4B  and C).Collectively, our data suggest that HSV-1 infection enhances gliosis and microglial activation in 5xFAD mice.

HSV-1 infection induces enhanced microglial phagocytosis of HSV-GFP-positive neuronal cells
The production and aggregation of Aβ are attributed to the imbalance between Aβ production and clearance [44].Having observed significant Aβ plaque deposition in 5xFAD mice after HSV-1 infection, we next investigated whether APP processing was involved in Aβ production.To test this hypothesis, we examined a series of key factors involved in Aβ production.In comparison to 5xFAD control mice, there was no significant change in the expression levels of APP, PS1, or the APP processing secretases ADAM10 and BACE1 in the HSV-infected 5xFAD mice (Figure S1).These results demonstrate that HSV-1 infection exacerbates Aβ burden without altering APP expression and processing, both in young and aged 5xFAD mice.
Given the significant accumulation of Aβ plaques with unchanged Aβ production in 5xFAD mice after HSV-1 infection, we next sought to explore whether HSV-1 infection affects Aβ clearance.Since microglia play a crucial role in phagocytizing and degrading extracellular Aβ in the brain of AD mice, we then performed immunostaining assay using anti-Aβ antibody along with anti-Iba1 antibody to visualize the colocalization of Aβ and microglia.Confocal images showed that microglia clustered around the Aβ plaques in 5xFAD-Ctrl mice (Fig. 5A).However, we observed a significant decrease in the number of Iba1 + microglia near Aβ plaques and a significant increase in the number of Iba1 + microglia near the viral core in 5xFAD mice following HSV-1 infection (Fig. 5A and B).
To further elucidate whether the enhanced recruitment of microglia around the viral core affects Aβ engulfment and clearance, we performed immunostaining of CD68, a phagocytic marker for microglia.Compared with the control group, HSV-1 infection markedly upregulated CD68 expression in all age groups of 5xFAD mice (Fig. 5C and D, Figure S2A and S2B).Quantitative analysis of the colocalization area of CD68 with Aβ or HSV-GFP revealed a significant increase in GFP + CD68 + microglia and a significant decrease in Aβ + CD68 + microglia in 5xFAD-HSV mice (Fig. 5E-G, Figure S2C), This suggests that microglia preferentially phagocytize HSV-GFP-positive neuronal cells after HSV-1 infection, but not Aβ plaques, leading to Aβ accumulation in 5xFAD-HSV mice.To further characterize this microglia-HSV-1 module, we examined the effects of HSV-1 infection on Aβ uptake in cultured BV2 and primary microglial cells by flow cytometry.BV2 and primary microglial cells were incubated with Alexa 555-labeled Aβ 1-42 or Alexa 555-labeled Aβ 1-42 plus HSV-1-GFP from HT-22 neuronal cells for 4 h.Consistent with our in vivo data, we observed a decrease in Aβ 1-42 uptake and an increase in GFP-positive phagocytosis upon incubation with GFPpositive cell debris (Fig. 6A, B, Figure S3A-B).In addition, we confirmed this phenomenon using an immunostaining assay, which consistently showed that BV2 and primary microglial cells preferred to phagocytize GFPpositive cell debris (Fig. 6C-E, Figure S3C-D).Together, these results suggest that HSV-1 infection enhances the recruitment of microglia to the viral core and decreases their phagocytic uptake of Aβ plaques, consequently leading to Aβ accumulation and deposition both in vivo and in vitro.

Microglia are indispensable to control Aβ plaque aggregation after HSV-1 infection
Our results indicated that microglia may be not able to perform normal Aβ clearance following HSV-1 infection.To further confirm the role of microglia in Aβ pathology, we employed the CSF1R inhibitor, PLX3397, to deplete microglia [45,46].5xFAD mice were fed with PLX3397-formulated diet for 14 days and then injected with PBS or HSV-GFP for another 7 days (Fig. 7A).After PLX3397 treatment, both microglia numbers and their phagocytic ability, as assessed by Iba1 and CD68 staining, were substantially reduced in both 5xFAD-Ctrl mice and 5xFAD-HSV mice (Fig. 7B-E, Figure S4).To determine if microglia are responsible for HSV-1-mediated plaque aggregation, we depleted microglia with PLX3397, followed by the administration of HSV-1 to the hippocampus.Consistent with the previous findings, treatment of 5xFAD mice with PLX3397 led to a reduction in Aβ plaque deposition (Fig. 7F, G and Figure S5).However, compared to 5xFAD-HSV mice in the absence of PLX3397 treatment, the plaque load was markedly increased in 5xFAD-HSV mice treated with PLX3397 (Fig. 7F, G, Figure S5), indicating that microglia play an indispensable role in controlling HSV-1-mediated Aβ plaque aggregation.Furthermore, we examined the protein amounts of Aβ in 5xFAD mice treated with PLX3397 and injected with HSV-1 and found that the amounts of Aβ were significantly increased in 5xFAD-HSV mice treated with PLX3397 (Fig. 7H, I).Together, these results indicate that microglia play an indispensable role in maintain the homeostasis of HSV-1-GFP and Aβ plaque aggregation in 5xFAD-HSV mice.

NLRP3 inflammasome is activated after HSV-1 infection in 5xFAD mice
Our data suggested that depletion of microglia did not allow to establish a precise role for Aβ deposition and HSV-1 infection in AD pathogenesis.Neuroinflammation, an important hallmark of AD, is associated with Aβ deposition and pathogen invasion [47][48][49].To further define the regulation mechanism of HSV-1 and Aβ, we characterized the innate immune signaling pathways induced by HSV-1.Recent reports support the hypothesis that cGAS-STING pathway or NLRP3 inflammasome signaling may act as a driver of neuroinflammation in the brain [50,51].To investigate whether the cGAS-STING pathway or the NLRP3 inflammasome pathway is involved in HSV-1-mediated AD pathology, we injected HSV-1 virus into the brains of 5xFAD mice for the indicated time periods and found that HSV-1 infection promotes Aβ accumulation at day 3 and day 7 (Fig. 8A and  B), consistent with our findings in Fig. 1.
Next, we examined the protein markers of signaling cascades of NLRP3 inflammasome and the cGAS-STING pathway in the hippocampus of 5xFAD mice for the indicated time periods.We observed that phosphorylation levels of TBK1 and STING were upregulated in the early stages (6 h-24 h) following HSV-1 infection (Fig. 8C and  D).Additionally, NLRP3 and cleaved-caspase1 protein expression levels were upregulated on day 3 and day 7 after HSV-1 infection (Fig. 8C and D).Further immunostaining assay confirmed that the NLRP3 inflammasome was activated in the hippocampus of 3-month-old 5xFAD mice at day 7 after HSV-1 infection (Fig. 8E).This phenomenon was also observed in 1-month-old 5xFAD mice and 6-month-old 5xFAD mice at day 7 after HSV-1 infection (Figure S6).Subsequently, IL-1β production was also assessed by ELISA assay in 1-, 3-, and 6-monthold 5xFAD mice infected with PBS or HSV-GFP.We observed increased IL-1β levels in 1-, 3-, and 6-monthold 5xFAD mice after HSV-1 infection (Figure S6C, S6E and S6H).
We then performed immunofluorescence double staining of NLRP3 along with the microglia marker Iba1, astrocyte marker GFAP, and observed that NLRP3 is mostly expressed in microglia and to a lesser extent in GFP-positive neuronal cells (Fig. 8F and Figure S7A-B).In addition, we detected significantly more ASC specks in 5xFAD mice after HSV-1 infection (Fig. 8G), (See figure on next page.)Fig. 3 HSV-1 infection leads to gliosis in 5xFAD mice.A Representative images of microglia (Iba1, red) and astrocytes (GFAP, magenta) in the hippocampus of 1-month-old 5xFAD mice seven days after HSV-GFP infection.Scale bars, 20 μm.B Quantification of Iba1 + microglia and GFAP + astrocytes in the hippocampus of 1-month-old 5xFAD mice seven days after HSV-GFP infection.(n = 5 mice per group).C Representative images of microglia (Iba1, red) and astrocytes (GFAP, magenta) in the hippocampus of 3-month-old 5xFAD mice seven days after HSV-GFP infection.Scale bars, 20 μm.D Quantification of Iba1 + microglia and GFAP + astrocytes in the hippocampus of 3-month-old 5xFAD mice seven days after HSV-GFP infection.(n = 5 mice per group).E Representative images of microglia (Iba1, red) and astrocytes (GFAP, magenta) in the hippocampus of 6-month-old 5xFAD mice seven days after HSV-GFP infection.Scale bars, 20 μm.F Quantification of Iba1 + microglia and GFAP + astrocytes in the hippocampus of 6-month-old 5xFAD mice seven days after HSV-GFP infection.(n = 5 mice per group).All data are presented as means ± SEM. *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001 primarily expressed in microglia (Figure S7C) and responding to Aβ accumulation after HSV-1 infection (Figure S8).These results indicate that the expression pattern of the NLRP3 inflammasome is consistent with Aβ accumulation.Together, our data show that the NLRP3 inflammasome is activated after HSV-1 infection, and its expression trend is consistent with Aβ accumulation.Intracranial HSV-1 infection disrupts the blood-brain barrier (BBB) and leads to the infiltration of periphery immune cells [52,53].Macrophages and T cells infiltrate and play a variety of key roles in the immune defense system in neurodegenerative diseases, CNS injury, and CNS infections.Activated microglia, macrophages, and T cells secrete a series of cytokines (e.g., IL-6, IL-1β, TNFα, and IL-10) and chemokines (e.g., CCL5, CCL2, and MCP-1) to exert different functions [54][55][56][57][58].To investigate the inflammatory responses after HSV-1 intracranial infection, we performed immunostaining assays using anti-CD3 and anti-F4/80 antibodies along with anti-Iba1 antibody and observed a significant infiltration of T cells and macrophages (Figure S9A-D).Subsequently, we isolated total RNA from the non-infected and HSV-infected hippocampi of 3-month-old 5xFAD mice three or seven days after HSV-1 infection.The results showed that inflammatory genes significantly increased three days after HSV-1 infection (Figure S9E-F).Overall, our data reveal that macrophages and T cells infiltrate and secrete inflammatory factors after HSV-1 infection in 5xFAD mice.

Blockade of NLRP3 inflammasome signaling reduces Aβ deposition and ameliorates cognitive deficits in 5xFAD mice after HSV-1 infection
Our data support a positive correlation between NLRP3 inflammasome activation and Aβ accumulation following HSV-1 infection.To further define if the NLRP3 inflammasome signaling drives Aβ accumulation and AD pathology after HSV-1 infection, we administrated MCC950 sodium, a selective small-molecular inhibitor of the NLRP3 inflammasome, to inhibit inflammasome activation [59,60].5xFAD mice received MCC950 sodium via intraperitoneal injection every second day for 21 days after HSV-1 infection (Fig. 9A).First, we examined the expression levels of the NLRP3 inflammasome through western blot and immunostaining assay and observed a significant inhibition of NLRP3 inflammasome activity after MCC950 treatment following HSV-1 infection (Fig. 9B-E).Next, we evaluated the effects of MCC950 treatment on Aβ deposition.We found that MCC950 treatment significantly reduced the total Aβ burden in 5xFAD mice after HSV-1 infection (Fig. 10A-C).We further examined the Aβ expression amounts by immunoblotting assay.Consistent with the decreased Aβ deposition in imaging data, the expression amounts of Aβ were also decreased in 5xFAD mice treated with MCC950 after HSV-1 infection (Fig. 10D and E).To further investigate whether inflammasome inhibition could affect uptake of HSV-1-infected cells as well as Aβ, we performed an immunostaining assay using an anti-CD68 antibody along with an anti-Aβ antibody to visualize the colocalization of Aβ, HSV-GFP and CD68.Consistent with our data in Fig. 5, microglia preferentially phagocytize HSV-GFP-positive cells after HSV-1 infection, but not Aβ plaques.However, MCC950 treatment did not affect the phagocytic preference of microglia for Aβ or HSV-GFP-positive cells (Figure S10).Overall, inhibition of NLRP3 inflammasome activity did not alter the phagocytic preference of microglia but reduced Aβ deposition.
Then, we assessed the social learning and memory of mice through Y-maze and Morris water-maze tests.Compared with 5xFAD control mice, 5xFAD-HSV mice showed a reduction in spontaneous alterations in the Y-maze test and were less efficient at finding the hidden platform (Fig. 10F-H), indicating a deficit in social learning and memory after HSV-1 infection.However, MCC950 treatment significantly reversed these deficits in 5xFAD mice after HSV-1 infection (Fig. 10F-H).The improvement of these phenotype upon MCC950 treatment was not affected by the total number of arm entries (Fig. 10F) and the swimming speed of mice (Fig. 10H).Collectively, these results suggest that MCC950 treatment to inhibit NLRP3 signaling can ameliorate AD pathology, including Aβ deposition and impaired cognitive function.

Discussion
AD is the most common neurodegenerative disorder in the world, and HSV-1 is one of the most common DNA viruses globally.In this study, we characterized the role and regulation mechanism of HSV-1 in 5xFAD mice.Microglia are essential in the modulatory network of HSV-1 infection and Aβ deposition.In addition, we combined viral infection, immunology, biochemistry, cell imaging, and animal approaches to confirm for the first time that HSV-1 infection induced the activation of NLRP3 inflammasome signaling to drive Aβ deposition and AD pathology (Fig. 11).Our results suggest that NLRP3 inflammasome may drive the development of AD in the HSV-1 infection condition, and inhibition of NLRP3 signaling have potential to prevent Aβ accumulation and cognitive decline of AD.
Despite considerable efforts, elucidating the pathogenesis of AD is challenging due to its complex and undefined etiology.Growing evidence implicates that viral or microbial infection of the central nervous system (CNS) are potential pathological factors in the etiology of sporadic AD.The AD pathogen hypothesis suggests that various pathogens act as triggers, initiating a pathological cascade that leads to the accumulation of Aβ [11][12][13][14].According to this hypothesis, Aβ peptides have been shown to possess antimicrobial and antiviral effects and are produced by the CNS as a defense mechanism.Viral or microbial infection result in rapid seeding and accelerated β-amyloid deposition [13].In this process, these pathogens also activate glial cells and induce a pro-inflammatory innate immune response, ultimately leading to progressive neurodegeneration and dementia [61].However, the precise mechanism between viral infections and AD remains unclear.In this study, we utilized the approach introduced by Eimer and coauthors [13].We administrated HSV-1-GFP into the hippocampus of 1-monthold, 3-month-old and 6-month-old 5xFAD mice and observed the aggregation and deposition of Aβ in the whole brain following HSV-1 infection.Moreover, we also observed a significant accelerated cognitive dysfunction in 3-month-old 5xFAD mice following HSV-1 infection.These results are consistent with the previous reports in HSV-1-induced Aβ deposition and suggest HSV-1 infection leads to Aβ deposition and cognitive deficits in AD mouse model.Microglia are the resident innate immune cells in the CNS and have been shown to play major roles in internalization and degradation of Aβ [62].They are the primary phagocytes in the CNS for uptake and proteolytic clearance of both soluble and fibrillary forms of Aβ [63].Inflammatory responses influence the activation status of microglia and subsequently regulate their ability to uptake and degrade Aβ [63].HSV-1 brain infection leads to persistent activation of microglia, which results in the upregulated expression of type I interferon (IFN) in a cGAS-STING-dependent manner to exert antiviral function [49,64].However, abnormal activation of microglia may also lead to dysregulated microglia function, including phagocytosis and inflammation [42,[65][66][67].Here, we observed that microglia were activated after HSV-1 infection.In the viral core, microglia often displayed an activated "rounded" and "amoeboid" morphology characterized by shorter processes, larger cell bodies and reduced microglia volumes.Interestingly, we observed that microglia were recruited to the viral core and engulfed HSV-1-GFP-positive neuronal cells, demonstrated by CD68 and Aβ coimmunostaining.Our results demonstrate that the recruitment of microglia to the viral core increased the phagocytic ability to viruses following HSV-1 infection.In this case, no enough microglia would be recruited to clear Aβ, causing Aβ deposition.Our results indicate that microglia modulate their phagocytic preferences, enhancing their uptake of viruses after HSV-1 infection and reducing Aβ uptake, ultimately leading to Aβ accumulation and deposition.In recent years, with the fast development of techniques, growing research on microglia has remarkably revealed their phagocytic roles in neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), frontotemporal dementia (FTD), and amyotrophic lateral sclerosis (ALS) [37,[68][69][70].Microglia have a high phagocytic capacity and can clear pathological protein aggregates (Aβ, α-synuclein, and TDP43) [71].Nevertheless, excessive extracellular protein aggregate release and autophagy impairment both contribute to pathological progression and neurodegeneration.Thus, enhancing microglial phagocytosis is considered a promising strategy for the therapy of neurodegenerative diseases.
Microglia execute their roles as the first line of defense against CNS infection through their immune response mediated by immune receptors [72,73], including NLRmediated responses such as the NLRP3 inflammasome [35,36], and nucleic acids receptor-mediated responses, such as RIG-1 and cGAS [34,38,74].Previous studies have found that NLRP3 is activated in AD mouse models and AD patients, contributing to the progression of AD pathology.NLRP3 inflammasome deficiency resulted in decreased deposition of Aβ and reduced tau hyperphosphorylation, ultimately delaying the progression of AD pathology [51,75].In this study, we observed that the NLRP3 inflammasome signaling was activated on day 3 and 7 after HSV-1 infection, with its activation trend being correlated with Aβ accumulation.Therefore, we speculated that the NLRP3 inflammasome was involved in the accumulation of Aβ.To testify this hypothesis, we employed a selective small-molecular inhibitor (MCC950) of the NLRP3 inflammasome signaling to inhibit inflammasome activation and revealed that MCC950 treatment significantly prevented Aβ deposition and alleviated the cognitive impairments induced by HSV-1 infection.These findings suggested that NLRP3 inflammasome activation mediates HSV-1-induced Aβ pathology in 5xFAD mice.Other selective NLRP3 inflammasome inhibitors, such as OLT1177 (Dapansutrile), exert therapeutic effects in the experimental autoimmune encephalopathy (EAE) mouse model and attenuate the infiltration of CD4 T cells [76].Stavudine (D4T), stimulates Aβ phagocytosis by macrophages to eliminate Aβ [77].Overall, previous studies and our work indicate that patients with AD and HSV-1 infection may potentially benefit from NLRP3-targetted treatment strategies.
As a typical DNA virus, HSV-1 could activate cGAS-STING DNA sensing pathway [38,78].In addition, previous study reveals that HSV-1 infection could induce releases of mitochondrial DNA (mtDNA) into cytosol to activate cGAS-STING signaling.Indeed, we detected strong activation of cGAS-STING signaling at early stage after HSV-1 infection in this study.Recent studies show that abnormal activation of cGAS-STING signaling pathway is a driven-factor of aging and neurodegeneration [79].Of note, the aberrant cGAS-STING activation are also involved in AD [80][81][82][83].The mtDNA release into cytosol of the microglial could activate cGAS-STING signaling to contribute to AD pathogenesis in 5xFAD mice [81].Tau can also activate cGAS-STING pathway by inducing microglia mtDNA leakage [82].Consistently, cGAS deletion or inhibitors of cGAS (or STING inhibitor) could restrict cGAS-STING pathway and mitigate AD progress in different AD disease models [80,81].Therefore, we hypothesize that, in AD, distinct sources of innate immune components can be activated in different time course to promote neuroinflammation.We propose that distinct innate immune signaling pathways, including cGAS-STING pathway and NLRP3 inflammasome, may contribute to neuroinflammatory phenomena and AD pathogenesis with distinct mechanisms.Further studies are required to investigate how these innate immune pathways are regulated and cooperates with each other during the pathological phenomena of AD.
Aβ proteins, herpesviruses, and innate immunity are all ancient (at least 300 million years old).Understanding the underlying regulatory mechanisms in brain and neurodegenerative disorders would be very interesting and valuable.While previous reports have suggested a potential link between HSV-1 and Aβ accumulation through the use of AD mouse model and 3D human brain model [11,13,67,84], the cognitive impairment and the precise modulatory mechanism behind it remain unclear.In this study, we demonstrate that HSV-1 infection leads to Aβ deposition, activation of NLRP3 inflammasome, and accelerated cognitive deficits in 5xFAD mice.Our data reveal that microglia and the NLRP3 inflammasome play crucial role in AD pathology induced by HSV-1 infection.Our results provide some information to unravel the mechanism of HSV-1-induced AD pathogenesis, suggesting new therapeutic approaches for this multifactorial and devastating neurodegenerative disease.

Fig. 2 Fig. 2 (
Fig. 2 HSV-1 infection exacerbates Aβ deposition in 1-month-old and 6-month-old 5xFAD mice.A and B Representative images (A) and quantification of Aβ plaques and plaque area (B) in the whole brain and hippocampus of 1-month-old 5xFAD mice seven days after HSV-GFP infection.Scale bars, 200 μm, 100 μm and 50 μm.(n = 5 mice per group).C and D Representative images (C) and quantification of Aβ plaques and plaque area (D) in the whole brain of 6-month-old 5xFAD mice seven days after HSV-GFP infection.Scale bars, 200 μm, 100 μm and 50 μm.(n = 5 mice per group).E Representative images (E) and quantification of Aβ plaques and plaque area (F) in the whole brain of 6-month-old 5xFAD mice seven days after HSV-GFP infection.Scale bars, 200 μm, 100 μm and 50 μm.(n = 5 mice per group).G Immunoblotting and quantification of Aβ protein levels in the hippocampus of 1-month-old 5xFAD mice (Ctrl), 5xFAD mice infected with PBS (Vehicle) or 5xFAD mice seven days after HSV-GFP infection.(n = 3 mice per group).H Immunoblotting and quantification of Aβ protein levels in the hippocampus of 6-month-old 5xFAD mice (Ctrl), 5xFAD mice infected with PBS (Vehicle) or 5xFAD mice seven days after HSV-GFP infection.(n = 3 mice per group).All data are presented as means ± SEM. n.s.: p > 0.05, *p ≤ 0.05, **p ≤ 0.01 (See figure on next page.)

Fig. 6
Fig. 6 HSV-1 infection induces enhanced microglial phagocytosis of HSV-GFP-positive neuronal cells in vitro.A Representative FACS dot plots of engulfed GFP-positive cell fragments or 555-labed Aβ in BV2 microglial cells are shown.These experiments are repeated three times.B Quantification of engulfed GFP-positive cell fragments or 555-labed Aβ in the BV2 microglial cells as measured by flow cytometry.C Representative images of microglial phagocytosis of GFP-positive cell fragments or 555-labed Aβ after uptake for 4 h in cultured BV2 microglial cells.These experiments are repeated three times.Scale bars; 20 μm.D Representative images of microglial phagocytosis of GFP-positive cell fragments or 555-labed Aβ after uptake for 4 h in cultured BV2 microglial cells.These experiments are repeated three times.Scale bars; 5 μm.E Quantification of internalized 555-labed Aβ using ImageJ software.(n = 10 per group).All data are presented as means ± SEM. **p ≤ 0.01, ****p ≤ 0.0001

(Fig. 7
See figure on next page.)Depletion of microglia increases Aβ plaque deposition and reduces microglial phagocytosis after HSV-1 infection.A Scheme for PLX3397 administration and HSV-GFP injection in 5xFAD mice.B and C Representative images (B) and quantification of Iba1-positive microglial cells (C) in the hippocampus of 3-month-old 5xFAD mice seven days after HSV-GFP infection following 21 days treatment with PLX3397.Scale bars, 50 μm.(n = 5 mice per group).D and E Representative images (D) and quantification of CD68-positive microglial cells (E) in the hippocampus of 3-month-old 5xFAD mice seven days after HSV-GFP infection following 21 days treatment with PLX3397.Scale bars, 50 μm.(n = 5 mice per group).F and G Representative images (F) and quantification of Aβ plaques and plaque area (G) in the whole brain of 3-month-old 5xFAD mice seven days after HSV-GFP infection following 21 days treatment with PLX3397.Scale bars, 200 μm, 100 μm and 50 μm.(n = 5 mice per group).H and I Immunoblotting and quantification of Aβ protein in the hippocampus of 3-month-old 5xFAD mice seven days after HSV-GFP infection following 21 days treatment with PLX3397.Data are presented as means ± SEM. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001

Fig. 8 Fig. 8 (Fig. 9 Fig. 10 Fig. 10 (
Fig. 8 NLRP3 inflammasome is activated after HSV-1 infection in 5xFAD mice.A Representative images of Aβ (red) and Thio-S (gray) in the hippocampus of 3-month-old 5xFAD mice infected with PBS or HSV-GFP over the indicated time periods.B Quantification of Aβ plaques in the hippocampus of 3-month-old 5xFAD mice infected with PBS or HSV-GFP over the indicated time periods.(n = 5 mice per group).C Western blot analysis of NLPR3 inflammasome protein markers and cGAS-STING signaling pathway protein markers from the hippocampi over the indicated time periods upon HSV-GFP infection.D Quantification of the band intensity in C using ImageJ software.(n = 3 mice per group).E Representative images and quantification of NLRP3-positive cells (magenta) in the hippocampus of 3-month-old 5xFAD mice seven days after HSV-GFP infection.Original magnification × 40, scale bars; 20 μm.Zoom-in images with a scale bar equal to 10 μm.(n = 5 mice per group).F Representative images of NLRP3 (gray) co-stained with microglia (Iba1, red) and astrocytes (GFAP, magenta) in the hippocampus of 3-month-old 5xFAD mice seven days after HSV-GFP infection.Scale bars; 14 μm.G Representative images and quantification of ASC-positive cells (magenta) in the hippocampus of 3-month-old 5xFAD mice seven days after HSV-GFP infection.Original magnification × 40, scale bars; 20 μm.Zoom-in images with a scale bar equal to 10 μm.(n = 5 mice per group).Data are presented as means ± SEM. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001 (See figure on next page.)

Fig. 11
Fig. 11 Schematic of HSV-1 infection accelerates the progression of Alzheimer's disease by modulating microglial phagocytosis and activating NLRP3 pathway